1. Field of the Invention
The invention relates to hybridoma cell lines and monoclonal antibodies produced therefrom which may be used to detect the bacterium Mycobacterium avium subspecies paratuberculosis. 
2. Description of the Prior Art
The genus Mycobacterium comprises a diverse group of animal and human pathogens as well as saprophytes, many of which are ubiquitous in the environment. Mycobacterium avium subsp. paratuberculosis is a member of the Mycobacterium avium complex (MAC) and an animal pathogen that is highlighted by the large financial burden that it places on the dairy industry due to Johne's disease (JD). Figures extrapolated from the 1996 NAHMS dairy survey suggest that the cost of this disease was over $200 million per year (Ott et al. 1999. Herd-level economic losses associated with Johne's disease on US dairy operations. Prey. Vet. Med. 40:179-192). The growing recognition of M. avium subsp. paratuberculosis infection in wildlife species is of considerable concern, since it may affect our ability to control or eradicate JD from domesticated animals (Corn et al. 2005. Isolation of Mycobacterium avium subsp. paratuberculosis from free-ranging birds and mammals on livestock premises. Appl. Environ. Microbiol. 71:6963-6967; Daniels et al. 2003. The risk of disease transmission to livestock posed by contamination of farm stored feed by wildlife excreta. Epidemiol. Infect. 130:561-568).
Despite the research difficulties and economic consequences of JD, very few reports have described specific, antigen-based detection reagents for M. avium subsp. paratuberculosis. With the exception of a single study published 10 years ago (Mutharia et al. 1997. Analysis of culture filtrate and cell wall-associated antigens of Mycobacterium paratuberculosis with monoclonal antibodies. Infect. Immun. 65:387-394), the scientific literature is silent on the subject of M. avium subsp. paratuberculosis monoclonal antibodies (MAbs) and their use in JD research. Very recently, single-chain antibodies were selected by cloning heavy and light chains from sheep with JD (Berger et al. 2006. Isolation of high-affinity single-chain antibodies against Mycobacterium avium subsp. paratuberculosis surface proteins from sheep with Johne's disease. Clin. Vaccine Immunol. 13:1022-1029). This effort has resulted in two very promising recombinant antibodies; however, the M. avium subsp. paratuberculosis proteins that these antibodies react with remain unknown. The overall lack of detection reagents for M. avium subsp. paratuberculosis is in stark contrast to the availability of detection reagents for other bacterial pathogens of cattle, such as Brucella or Mycobacterium bovis, for which scores of MAbs are available to researchers (Bowden et al. 1997. Rapid identification of rough Brucella isolates by a latex coagglutination assay with the 25-kilodalton outer membrane protein and rough-lipopolysaccharide-specific monoclonal antibodies. Clin. Diagn. Lab. Immunol. 4:611-614; Cloeckaert et al. 1996. Production and characterization of monoclonal antibodies to Brucella melitensis cytosoluble proteins that are able to differentiate antibody responses of infected sheep from Rev. 1 vaccinated sheep. J. Med. Microbiol. 45:206-213; Kuchinka et al. 1990. Production and partial characterization of monoclonal antibodies to the neotype strain of Mycobacterium bovis. Am. J. Vet. Res. 51:1608-1615; Mertens et al. 2001. Selection of phage-displayed peptides recognized by monoclonal antibodies directed against the lipopolysaccharide of Brucella. Int. Rev. Immunol. 20:181-199; Morris et al. 1985. The identification of antigenic determinants on Mycobacterium bovis using monoclonal antibodies. J. Gen. Microbiol. 131:1825-1831; and Weynants et al. 1997. Characterization of smooth lipopolysaccharides and O polysaccharides of Brucella species by competition binding assays with monoclonal antibodies. Infect. Immun. 65:1939-1943).
Against this background, recent changes have modified the JD research landscape. Within the United States, a national consortium, entitled the Johne's Disease Integrated Program (JDIP), has identified the high research priorities and the knowledge gaps necessary to combat JD. Similar JD research consortiums have also recently formed in Europe and New Zealand. One of the priorities identified by JDIP is the development of specific detection reagents such as MAbs for M. avium subsp. paratuberculosis. More than just their obvious application for the diagnosis of JD, MAbs are critical reagents in cell biology and pathogenesis studies, including studies of macrophage-pathogen interactions, studies that use Luminex and magnetic bead technologies, as well as histopathology studies. MAbs that detect specific M. avium subsp. paratuberculosis proteins are ideal for incorporation into diagnostic assays such as those already developed for Campylobacter (Brooks et al. 2004. Evaluation of a monoclonal antibody-based enzyme-linked immunosorbent assay for detection of Campylobacter fetus in bovine preputial washing and vaginal mucus samples. Vet. Microbiol. 103:77-84) and Escherichia coli (Kerr et al. 2001. Development of a monoclonal sandwich ELISA for the detection of animal and human Escherichia coli O157 strains. J. Appl. Microbiol. 90:543-549). Furthermore, MAbs have application in the histopathological examination of infected tissues, typically the lamina propria of the intestine, where acid-fast staining has historically been used to demonstrate the presence of M. avium subsp. paratuberculosis, albeit with a low sensitivity and specificity (Thoresen et al. 1994. Comparison of immunohistochemistry, acid-fast staining, and cultivation for detection of Mycobacterium paratuberculosis in goats. J. Vet. Diagn. Investig. 6:195-199).
In view of the above, the need remains for improved diagnostic reagents for detecting and identifying M. avium subsp. paratuberculosis. 